The expression of fusion proteins is well known in the art and it is disclosed for instance in the following patent publications: EP-6694, EP-20290, U.S. Pat. Nos. 4,898,830, 5,452,199, EP-213472, EP-196864, EP-461165.
The manufacture of recombinant proteins of interest (e.g., human) in suitable expression systems is one of the main industrial applications of recombinant DNA technology. If, as is often the case, the protein is unstable in the host cell, it may be advantageous to manufacture the protein of interest in the form of a fusion moiety comprising a protective (or stabilising) protein which will be subsequently processed at a specific predetermined site in order to free the desired protein.
Another reason for making fusion proteins is to increase expression levels and/or to facilitate the purification process by the selection of suitable polypeptide sequences and attachment of one or more of such sequences to the amino- or carboxy-terminal ends of the polypeptide or protein of interest. A further reason for expressing a fusion protein might be that of having the characterizing features of two or more different proteins or subunits in a single chain, thus providing a higher activity/dose ratio of the protein itself, and/or avoiding the extra steps to obtain ligation of two subunits. One of the classical problems associated with the expression of recombinant proteins is that of obtaining a valuable and reliable source of the nucleic acid to be expressed.
One way of addressing this problem is to use an mRNA coding for the protein to be expressed. mRNA is however not always easily found under natural conditions. For example, in the case of beta-chain human follicle stimulating hormone (FSH), the corresponding mRNA can be found only in human pituitary cells and only in minute quantities. To be useable, this mRNA must be taken from the human pituitary cells immediately after death.
An alternative is to obtain the coding sequences of interest from genomic DNA. This is however a cumbersome and time consuming process as often, large amounts of unwanted DNA material are present in the initial sample which increases the probability of mutations and other errors. The need therefore still exists for an improved process for generating nucleic acid sequences to be used for expressing polypeptides, and in particular for generating nucleic acid sequences to be used for expression of heterologous recombinant fusion polypeptides.